The present invention relates to an improved process for obtaining insulins or insulin derivatives with correctly linked cystine bridges in the presence of cysteine or cysteine hydrochloride and of a chaotropic auxiliary compound, with folding being carried out in a reaction mixture in which the volume-to-surface ratio is greater than 1 and/or the oxygen concentration is 1-15 mg/l.
Human insulin is a protein with two amino acid chains of a combined 51 amino acid residues. The two amino acid chains contain 6 cysteine residues, with two cysteine residues being linked to one another via a disulfide bridge. In biologically active human insulin, the A and B chains are linked to one another via two cystine bridges and a further cystine bridge is present in the A chain. Statistically, 15 disulfide bridge formations are possible in one human insulin molecule. Only one of these 15 possible formations occurs in biologically active human insulin. The following cysteine residues are linked to one another in human insulin:    A 6-A 11    A 7-B 7    A 20-B 19The letters A and B represent the respective insulin amino acid chain, and the number indicates the position of the amino acid residue, which is counted from the amino end to the carboxyl end of the particular amino acid chain. Disulfide bridges may also form randomly between any two human insulin molecules, enabling a vast number of different disulfide bridges to be produced easily.
A known process for preparing human insulin is based on the use of human proinsulin. Human proinsulin is a protein with a linear amino acid chain of 86 amino acid residues, in which the B and A chains of human insulin are linked to one another via a C peptide bridge containing 35 amino acid residues. The disulfide bridges present in human insulin are formed via an intermediate, with the cysteine residues of human insulin having a sulfur protective group, for example an S-sulfonate group (—S—SO3−: see EP 0 037 255). Another known process is one for obtaining proinsulin with correctly linked cystine bridges (Biochemistry, 60, (1968), pages 622 to 629) that starts from proinsulin obtained from pig pancreas in which the cysteine residues are present as thiol residues (—SH). The term “correctly linked cystine bridges” means the disulfide bridges which occur in biologically active mammalian insulin.
Genetic engineering processes allow precursors of insulin or insulin derivatives, in particular human proinsulin or proinsulin whose amino acid sequence and/or amino acid chain length deviate from human insulin, to be prepared in microorganisms. The proinsulins produced by genetically altered Escherichia coli cells do not have any correctly linked cystine bridges. One process for obtaining human insulin by using E. coli (EP 0 055 945) is based on the following steps:
Fermentation of microorganisms—disruption of cells—isolation of fusion protein—cyanogen halide cleavage of fusion protein—isolation of cleavage product having the proinsulin sequence—protection of proinsulin cysteine residues by S-sulfonate groups—chromatographic purification of S-sulfonate—formation of correctly linked cystine bridges—desalting of proinsulin—chromatographic purification of proinsulin with correctly linked cystine bridges—concentration of proinsulin solution—chromatographic purification of concentrated proinsulin solution—enzymatic cleavage of proinsulin to obtain human insulin—chromatographic purification of obtained human insulin.
Disadvantages of this process are the number of steps and the losses during the purification steps, resulting in a low insulin yield. Due to the multi-stage process route, considerable losses must be accepted. From the isolated fusion protein stage via cyanogen halide cleavage, sulfitolysis and purification of proinsulin, losses of up to 40% of proinsulin can be expected (EP 0 055 945). Losses of a similar size may occur during the subsequent purification steps up to the final product.
Increased yields can be obtained in the genetically engineered production of human insulin or insulin derivatives, if the number of steps required can be reduced substantially.
U.S. Pat. No. 5,473,049 to Obermeier et. al. (EP 0 600 372 A1) and EP 0 668 292 A2 disclose a correspondingly improved process for obtaining insulin and insulin derivatives, which involves converting the insulin precursor or insulin derivative precursor, whose cystine bridges are not correctly linked, to an insulin precursor or insulin derivative precursor, which has correctly linked cystine bridges, in the presence of a mercaptan, for example cysteine, and of at least one chaotropic auxiliary compound, for example urea or guanidine hydrochloride. The disclosed process comprises first dissolving said proteins in aqueous solutions of a chaotropic auxiliary compound or of mixtures of various chaotropic auxiliary compounds at a very low concentration. The protein mixture is then mixed with an aqueous mercaptan solution.